Lab 1

Microbiology Lab Safety

Lab 2 Respiration

Lab 3

Introduction to Culturing and Aseptic Technique

Good Lab Techniques

If you are allergic to nitrile gloves, please contact us and we will send you an alternative.

Please note that the times listed are approximations and may differ. Please read
through the procedure and plan accordingly.

Lab 1 Microbiology Lab Safety

Time Required: 30 minutes of prep time plus 2 - 3 days for observation
Additional Materials: Warm water and a stopwatch.

Lab 2 Respiration
Time Required: 60 minutes of prep time plus 3 hours for observation
Additional Materials: Water, stopwatch, permanent marker, and paper towels.

Lab 3 Introduction to Culturing and Aseptic Technique

Time Required: 60 - 90 minutes of prep time plus 6 days for observation (3 days per
experiment)
Additional Materials: Microwave or boiling water bath and a permanent marker.

eScience Labs, LLC designs every kit with safety as our top priority. Nonetheless, these are science
kits and contain items which must be handled with care.
Safety in the laboratory always comes first!
Always follow the instructions in your laboratory manual and these general rules:

Please thoroughly read the lab exercise before starting!

If you have any doubt as to what you are supposed to be doing and how to do it safe-

ly, please STOP and then:

Double-check the manual instructions.

Check www.esciencelabs.com for updates and tips.

Contact us for technical support by phone at 1-888-ESL-Kits (1-888-375-5487) or

by email at Help@esciencelabs.com.

Read and understand all labels on chemicals.

If you have any questions or concerns, refer to the Material Safely Data Sheets
(MSDS) available at www.esciencelabs.com. The MSDS lists the dangers, storage requirements, exposure treatment and disposal instructions for each chemical.

Consult your physician if you are pregnant, allergic to chemicals, or have other medical conditions that may require additional protective measures.

Remove all loose clothing (jackets, sweatshirts, etc.) and always wear closed-toe
shoes.

Long hair should be pulled back and secured and all jewelry (rings, watches, necklaces, earrings, bracelets, etc.) should be removed.

Safety glasses or goggles should be worn at all times. In addition, wearing soft contact lenses while conducting experiments is discouraged, as they can absorb potentially harmful chemicals.

When handling chemicals, always wear the protective goggles, gloves, and apron
provided.

Do not eat, drink, chew gum, apply cosmetics or smoke while conducting an experiment.

Work in a well ventilated area and monitor experiments at all times, unless instructed otherwise.

When working with chemicals:

Never return unused chemicals to their original container to avoid contamination.

Never place chemicals in an unmarked container to avoid identification or proper

disposal problems.

Always put lids back onto chemicals immediately after use to avoid contamination or potential hydration problems.

Never ingest chemicals. If this occurs, seek immediate help.

Call 911 or Poison Control 1-800-222-1222

Never pipette anything by mouth.

Never leave a heat source unattended.

If there is a fire, evacuate the room immediately and dial 911.

If a spill occurs, consult the MSDS to determine how to clean it up.

Never pick up broken glassware with your hands. Use a broom and a dustpan and
discard in a safe area.

Do not use any part of the lab kit as a container for food.

Safely dispose of chemicals. If there are any special requirements for disposal, it
will be noted in the lab manual.

When finished, wash hands and lab equipment thoroughly with soap and water.

Above all, use common sense! Read the manual carefully and pay close attention to the safety concerns prior to starting an experiment.

ESL Safety Video

ESL Scientific Method Video
How Big Is It?
How to Use a Microscope
Virtual Microscope
Spelling Bacteria Names Quiz
Measuring Volume Using a Graduated Cylinder
Unit Conversions
Sample Labware Image

Diffusion and Osmosis Tutorial

Passive Transport : Diffusion
The Cell: Passive Transport Diffusion
Enzymes: Docking Tutorial

Log on to the Student Portal

using these easy steps:
Visit our website,
www.esciencelabs.com, and click
on the green button (says
Register or Login) on the top
right side of the page. From
here, you will be taken to a login
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this is how you will enter the Student Portal for future visits. This
establishes your account with the
eScience Labs Student Portal.
Have fun!

Blood Agar and Hemolysis

Categories of Acquired Immunity
Components of a Prokaryote
Eukaryotic Cells: Assembling the Cell
Gram Stain Procedure
Correlation of Colony Morphology with Gram Stain Results
Identification of Lactose Positive and Lactose Negative Bacteria on MacConkey Agar
Mannitol Salt Agar

Urine Colony Counts

Gene Machine: The Lac Operon
DNA Isolation Virtual Lab
Microarray Virtual Lab

Stopwatch
Conversion Factors Tables

You will work with a variety of microorganisms throughout this lab kit. Cautious safety measures
have been put in place to ensure that your safety is maintained. Additionally, you are responsible
for carrying out proper disposal techniques at the end of each lab. Generally, this requires you to
flood any petri plates with a bleach solution and then sealing them with Parafilm .

Bleach should be purchased as soon as possible to ensure that you have it available at all times.
Bleach is available at most drug or grocery stores, and typically costs $2 - 4. You will need a minimum of 30 ounces of a 10% concentrated solution. If your solution has a higher bleach concentration, you can dilute the solution using water; just be sure to maintain a 10% concentration base.
Be sure to rinse the bleach down the drain with running water to avoid any plumbing concerns.

After bleaching, you will seal the plates or containers with Parafilm and dispose of them in the
trash. Standard trash receptacles are suitable for this, but please exercise caution if you live in an
environment with animals or children and ensure that they do not re-open or crack the containers.

The majority of the chemicals used in this kit can be safely disposed of down the drain. However,
there are several chemicals required for staining microorganisms that must be disposed of with
additional care. These chemicals include: Crystal Violet, Gram Decolorizer, and Safranin. These
chemicals are all classified by the EPA under code D001: Ignitable hazardous waste. This is due
to the alcohol concentration present in each solution. Local disposal requirements may vary.
Therefore, please contact your state hazardous waste authority to determine what steps are
needed to safely dispose of these chemicals. A library of each states contact information can be
found at the following website: http://www.epa.gov/osw/wyl/stateprograms.htm

A combination of alcohol immersion and candle flames are used for sterilization and staining purposes. You will need to purchase one bottle of isopropyl alcohol (rubbing alcohol) prior to starting
your lab experiments.

Isopropyl alcohol can typically be purchased at a grocery or drug store. One 16 ounce bottle is
sufficient. This will cost approximately $2.

Isopropyl alcohol may also be required as a chemical ingredient during some of your experiments.
You can use your purchased bottle of isopropyl alcohol for these procedures as well.

Alcohol bottles should always be placed away from open flames. If you have any questions about
the bleach or alcohol purchases please email eScience Labs at info@esciencelabs.com or call 888ESL-KITS.

Microbiologists must be extra diligent about using sterile techniques to ensure that equipment does
not become contaminated. In particular, petri dishe packages must be opened and closed with care
to prevent airborne contaminants from settling onto a dish. Therefore, you must seal the petri dish
packages with tape every time you close a package of dishes. More information about how to keep
a petri dish package sterile can be found by logging on to the eScience Labs Student Portal at
www.esciencelabs.com/portal and selecting the microbiology lab kit.

In addition to this, it is important to store your kit materials in the kit box. This further prevents any
surface or airborne contaminants from compromising your equipments sterility.

The MSDS for every chemical in this kit is provided at www.esciencelabs.com/educators/msds.

eScience Labs highly recommends that you download and print all of the MSDS prior to starting
your experiments. By doing so, you will ensure that you have all of the safety and cleanup information you need should you spill or encounter an accident during an experiment.

Thank you,
The eScience Labs Team

Lab 1

Microbiology Lab Safety

How to Safely Work With Microorganisms

Personal Protective Equipment

Terminology and Use of Experiment

Biological Safety Cabinets

Equipment

Welcome to the Introductory Microbiology Lab Manual!

Microbiology is the study of microorganisms. Microorganisms, or microbes, include any organism that is too
small to be seen by the unaided eye. Most of the microorganisms that we encounter are not harmful to us. In
fact, many are quite beneficial to us and our surrounding community. Microorganisms are used in industrial
production applications as well as employed to solve difficult environmental problems, such as oil spills.
Of course, there are a number of microorganisms that have harmful effects to humans, animals, plants, and
even other microorganisms! Some microbes cause people or animals to get sick; or, even have a destructive
effect on the environment. It is for this reason that there are defined safety procedures to follow and equipment to use when working with microorganisms.
Playing a game is more enjoyable than watching or reading about it. The same can be said for learning about
microbiology. Performing experiments using microorganisms that are common in our environment is fun and
informative. Throughout the experiments in this lab, you will come into contact with a variety of different microbes. While you will not be supplied with infectious organisms, you will be asked to isolate and culture
(grow) microbes from your surrounding environment. It is therefore critically important to learn how to properly work with microbes to ensure your safety.
This manual is written for performing microbiology experiments using
common microbes that can be readily isolated from every day materials,
such as soil, food, the bottoms of shoes, from inside the mouth, etc. Microbes from these sources are generally much less hazardous than
those that are used in a traditional microbiology lab setting. The experiments in this lab manual do not use dangerous human pathogens
(disease causing organisms). However, many microbes are potentially
dangerous, meaning that they would not normally cause disease in a Figure 1: The information provided
healthy individual but that they could cause disease if they were intro- in this lab will enable you to comduced (for example by an open wound, cut, or open mucus membrane) plete the microbiology labs in this
manual safely and successfully.

in an immunocompromised individual. Generally speaking, immunocompromised individuals are people

who have immune systems working at decreased or abnormal levels. This makes the person more susceptible to infections, disease, etc. If you have any concerns about your ability to perform these experiments safely, please consult your health care provider or instructor.
In addition to working with microorganisms, you will be working with chemicals, glassware, open flames,
and sharp objects that can be hazardous if used carelessly or improperly. You should always use the safety goggles, gloves, and other personal protective equipment provided in your kit.

Wash your hands with soap before beginning and after completing each lab.

Do not eat, drink, chew gum, or smoke while performing the experiments.

Do not put anything (pens, pencils, fingers, etc.) in your mouth while performing these experiments.

If you are performing these experiments in your home, prepare a work space that is free of any
food or drinks.

Disinfect your work area with isopropyl alcohol or 10% bleach solution both before and after
performing the experiments.

Extinguish flame(s) before using or spreading isopropyl alcohol.

Wear the safety equipment (see below) that is provided in the kit.

Always read the experiment thoroughly before beginning it.

Tie back long hair.

Use a broom and dustpan to collect broken glass, place it in newspaper, and dispose of properly. Never pick up broken glass with bare hands.

When using a flame source, ensure that the area is free of loose paper, gas, and any other
flammable or combustible material. Ensure that the flame is extinguished when you are finished
with it.

Material Safety Data Sheets (MSDS) for all chemicals provided are located on our website
(www.esciencelabs.com). These sheets contain all the necessary information regarding the
danger, safe use, and disposal of each chemical.

Safety Goggles/GlassesSafety goggles and glasses are used to protect your eyes and should be worn at all times while
doing these experiments, even if you are not actively working with microor ganisms.
GlovesGloves serve two purposes when working with microorganisms. Gloves protect your hands from
exposure to microorganisms and limit the spread of microorganisms present on your skin to the
cultures with which you are working. It is very important not to touch your work area with gloves
that have been contaminated with microbes. You should change your gloves before performing
other procedures. Gloves should always be worn while doing these experiments.
Lab Coat/ApronA lab coat or apron limits your exposure to the microbes
you are working with. It also limits exposing the cultures
you are working with to the microorganisms present on
your skin. You should wear a lab coat every time you perform an experiment.
Alternatively, a clean, long sleeved shirt that buttons or
snaps shut can be worn in place of a lab coat. The shirt
must fully cover your arms and be removable without
pulling it over your head.
Figure 2: Lab technician wearing the personal protective equipment required for
working with microorganisms. Note glassCollectively, safety goggles or glasses, gloves, and a lab es, gloves, and lab coat/apron. These
coat or apron comprise the personal protective equip- items should be worn when performing
ment that will be used in completing the experiments in the experiments in this lab manual.

this manual.
Safety ShowerSafety showers are used if someone/thing is exposed to a large quantity of microorganism(s). They
are also used if a hazardous chemical is spilled on a person and that person is unable to rinse it off
thoroughly in a sink. They may even be used in the event that clothes or an object catches fire. A
standard home shower sufficiently serves as a safety shower should the need arise.

Eye WashAn eye wash is used if your eyes are exposed to a microorganism or if a harmful chemical is
splashed into your eyes or face. In the home, use the nearest sink and flush your eyes or face with
cool water for at least 20 minutes. If the sink has a sprayer, use it to rinse your eyes or face while
making sure the water drains into the sink.
Fire ExtinguisherA fire extinguisher can be used to put out small to medium sized fires. Direct the fire extinguishing
material at the base of the fire to properly put it out. A home fire extinguisher should be rated for A, B,
and C type fires. This is clearly marked on the outside of the extinguisher.
Laboratory Fume HoodA laboratory fume hood removes harmful gases and fumes that may be present when doing an experiment. You should always use a fume hood when working with corrosive, noxious, or flammable
materials. Chemicals used in this kit will not require the use of a fume hood.
Biological Safety CabinetA biological safety cabinet (BSC) is used as the
main barrier to prevent exposure to infectious microorganisms in a laboratory setting. A BSC is not
the same as a fume hood. Airflow in a BSC is partially recirculated to the room in which a BSC is
housed. Additionally, a BSC contains a high efficiency particulate air (HEPA) filter which traps particulate (i.e., microorganisms), but not nonparticulate (i.e., fumes) matter. A BSC will not be
required to complete the experiments in this manuFigure 3: Biological Safety Cabinet.
al.

The Centers for Disease Control and Prevention (CDC), in cooperation with the National Institutes of Health
(NIH), have developed standards to offer protection when using biological pathogens. You are encouraged
to review the publication (available here: http://www.cdc.gov/biosafety/publications/bmbl5/index.htm) which
describes the practices, facilities, and safety equipment recommended for use in the four defined biosafety
levels (BSL). A BSL describes the minimum required containment measures that should be used to ensure
safety when working with various microorganisms. These measures are designed to reduce or eliminate
exposure to potentially infectious microbes. The four BSL are outlined in Table 1.
**Only BSL-1 procedures will be used in these experiments!!!**

Table 1: Biological Safety Levels

BSL

Agents

Practices

Equipment

Facilities

Not known to
cause disease in
health adults

Standard microbiological practices

None required

Open bench top

Hand washing sink

Associated with
disease; main hazard is percutaneous injury, ingestion, and mucus
membrane exposure

BSL-1 plus limited

access, biohazard
warning sign, sharps
precautions, manual
detailing decontamination and medical
surveillance polices

Class I or II BSC
for procedures
that generate
aerosols or
splashes;
gloves, lab coat,
goggles; respiratory protection if
needed

BSL-1 plus available

autoclave

Associated with
potential for aerosol transmission;
disease may be
lethal

BSL-2 plus controlled access; decontamination of all

waste and lab clothing; baseline serum
drawn for each worker

Class I or II BSC
for all procedures; gloves,
lab coat, goggles; respiratory
protection if
needed

BSL-2 plus physical

separation from access points, selfclosing, double door
access, no recirculated air, negative
airflow into BSL-3
lab

Associated with
risk of lifethreatening disease, aerosol
transmission or
unknown risk of
transmission

BSL-3 plus clothing

change before entering lab, shower when
leaving lab, all materials decontaminated

Class III BSC or

Class I or II if
used with full
body, independent air supply,
positive pressure suit

BSL-3 plus isolated

labs, dedicated air
supply and exhaust

Examples of BSL-1 Organisms: Escherichia coli (most species), Bacillus subtilus, and other organisms
commonly found in the local environment.
These microorganisms are well characterized and not known to cause disease in healthy adults. No special
laboratory equipment, including a BSC, is required. Work may be performed on an open bench and exposure is limited by the use of personal protective equipment: goggles, gloves, and a lab coat.
Examples of BSL-2 Organisms: Bacillus anthracis (anthrax), Bordetella pertussis (whooping cough), Clostridium botulinum (botulism), Human Immunodeficiency Virus (HIV).
Examples of BSL-3 Organisms: Mycobacterium tuberculosis (tuberculosis), Salmonella typhi (typhoid fever), Yellow Fever Virus.
Examples of BSL-4 Organisms: Smallpox virus, Ebola virus, hemorrhagic fever virus.

Again, you will NOT be using any organisms that require BSL-2, BSL-3, or BSL-4 containment procedures.
If you have any doubt or concerns about safety:
STOP!
Double-check the manual
Refer to our website: www.esciencelabs.com
Email: Help@esciencelabs.com
Call for help: 1-888-ESL-Kits (1-888-375-5487)
Contact your instructor

Microorganisms are tiny, typically only microns in length--much too small to be seen without a microscope.
Our bodies harbor bacteria that do not normally cause any disease (that is, they are not pathogenic), but
they can cause disease if transmitted to a person with an impaired immune system. Since they are so
small, it is difficult to know whether we have bacteria on our hands that we could easily transfer to another
person by direct contact (e.g., by shaking their hand) or indirectly (e.g., by touching a counter top which
someone else then touches). In this experiment, you will assess how easy it is to transfer a microorganism
with your hands, and how to prevent this transmission by hand washing. Baking yeast (Saccharomyces
cerevisiae) is an unicellular microorganism that does not cause disease but is an effective model for how
microorganisms can be easily transferred.

Prepared agar dishes should be stored upside-down in the refrigerator until used. This will pre-

Yeast packet

4 Disposable gloves

(1) 250 mL Beaker

Hot pad

Measuring spoon

Parafilm

1 tsp. Sugar

*10% Bleach solution

Nutrient agar

*Warm water

(4) 5 cm. Petri dishes

*Stopwatch

20 drops deionized water

*Permanent marker

4 Sterile transfer pipettes

*Paper towels

4 Sterile cotton swabs

Hand soap

*You must provide

vent condensation from disrupting the growing surface.

Before inoculating, let agar plate come to room temperature (about one hour).

After inoculating, replace the cover on the dish, seal with Parafilm, and store upside-down in a
warm location (not to exceed 37.7 C or 100 F).

You should see growth within a few days. The plate will start to smell once microorganisms are
growing.

Before disposing plates, kill the microorganisms by pouring bleach solution onto the agar surfaces and let sit for 20 minutes.

1. Loosen or remove the cap on the Nutrient agar bottle.

2. Loosen or remove the cap on the Nutrient agar bottle. Place the bottle in the microwave (if you do not
have a microwave, place the bottle in a heat-safe bowl and pour boiling water around the bottle) and
heat until the entire agar bottle is liquefied. You will need to remove the bottle and swirl every 10 seconds to distribute the heat.
Note: The bottle may explode, causing agar to project into your microwave if the bottle is not removed from heat every 10 seconds. It is very important to loosen or remove the cap to avoid creating a high pressure environment. If you notice the agar boiling over, STOP the microwave and let
the bottle cool down before handling. Hot agar can violently explode out of the bottle if heated too
quickly and/or shaken. After boiling has stopped, use a hot pad protecting your hands and remove
the bottle from the microwave. Use caution when removing the bottle from the microwave as it will
be HOT!
3. With a hot pad protecting your hands, remove the agar bottle from the microwave. Use caution when
removing the bottle from the microwave as it will be HOT!
4. Gently swirl the bottle a final time to mix the solution.
5. Pour the agar solution into the bottom half of four petri dishes.
Note: Approximately 5 mL should be enough agar to cover the entire bottom of the dish. If not, continue to add agar in approximately 1 mL increments until the bottom of the petri dish is completely
covered.
6. Place the lids onto the dishes and allow the agar to solidify undisturbed. This should take approximately
30 - 60 minutes. If you will not be using the dishes immediately, you can store them upside down in the
refrigerator after they have fully gelled. Remove them from the refrigerator and allow them to sit at room
temperature for at least one hour prior to use.

7. Use a permanent marker to label the bottom of the plates as #1, #2, #3, and #4.

8. Measure 230 mL of warm water into a 250 mL beaker.

9. Add 1 tsp. of sugar and the yeast packet to the beaker. Gently stir the solution to dissolve the ingredients. The solution should begin to foam/froth after a few minutes.

10. Put a disposable glove on your non-dominant hand (i.e., your left hand if you are right handed; your
right hand if you are left handed).
11. Use a disposable transfer pipette to add 8 - 10 drops of deionized water to the palm of your gloved
hand. Rub your hands together to spread the water over the surface of your hand. Remove and dispose
of the glove.
12. Wipe the surface of your dominant hand with the water on it with a clean cotton swab then gently rub
the cotton swab on the surface of nutrient agar plate #1. Put the lid on the plate and discard the cotton
swab.

13. Hold your hands under warm water.

14. Use soap and rub your hands together to form a visible lather. Continue rubbing for at least 20 seconds
(use your timer for this).
15. Rinse the soap off your hands, and dry them with clean paper towels.
16. Repeat Steps 10 - 11
17. Wipe the surface of your hand that had the water on it with a clean cotton swab then gently rub the cotton swab on the surface of nutrient agar plate #2. Put the lid on the plate and discard the cotton swab.
18. Repeat Steps 13 - 15.
19. Put a disposable glove on your non-dominant hand.
20. Use a disposable transfer pipette to add 8 - 10 drops of the yeast solution to the palm of your nondominant hand. Rub your hands together to spread the yeast over the surface of your hand. Remove
and dispose of the glove.
21. Wipe the surface of your hand with the yeast on it with a clean cotton swab then gently rub the cotton
ball on the surface of nutrient agar plate #3. Put the lid on the plate and discard the cotton swab.
22. Repeat Steps 13 - 15.

23. Wipe the surface of your dominant hand with a clean cotton swab then gently rub the cotton swab on
the surface of nutrient agar plate #4. Put the lid on the plate and discard the cotton swab.
24. Seal your plates with Parafilm, let them incubate in a warm location (not to exceed 37.7. C or 100 F)
and observe the growth. You should see colonies form in 25 days, depending on the temperature at
which the plates are incubated (the warmer the incubation temperature, the quicker the colonies will
form).
25. Count the number of colonies that grew and record the growth results in Table 1. Record growth as: (0)
= no growth; (+) = little growth (less than or equal to 5 colonies); (++) = moderate growth (6-20 colonies); (+++) = heavy growth (20 - 50 colonies); (++++) = confluent growth (no individual colonies, rather
they grow as a lawn and are uncountable).
26. Pour approximately 5 mL of the 10% bleach solution onto each surface of the remaining agar plates,
allowing it to cover the entire surface. Incubate them for 20 minutes, and then pour the bleach down
the sink with running water.
27. Seal the petri dishes with Parafilm and dispose of them in
the trash.

1. What constitutes personal protective equipment? When

should personal protective equipment be worn? Why is perFigure 4: Image of yeast colonies growing on culture media. Colonies should
be small, round, and white to slightly
yellow in color. Fuzzy appearing growth
2. Why are there increasing levels of biological containment pro- indicates mold contamination and
should not be counted.

sonal protective equipment important?

cedures? What level of biological containment will be necessary to complete the experiments in this manual?

Table 2: Experiment 1 Colony Growth

Plate #

Condition

Water minus hand washing

Water plus hand washing

Yeast minus hand washing

Yeast plus hand washing

Growth

3. What document should you refer to if you have questions regarding the safety or disposal of a chemical?
Where can this document be located?
4. List five safety procedures that should always be adhered to when working with microorganisms.
5. Which petri dish condition grew the most colonies? Which condition(s) grew the fewest colonies? What
can you conclude from these results?

Lab 3

Respiration

Cellular Energy

Anaerobic Respiration

Respiration

Aerobic Respiration

ATP is the energy currency of the cell. It is produced through a process called respiration. The energy molecules (ATP) generated through respiration, are available to fuel the processes of the cell as needed. When
ATP levels become too low a special protein signals the cell to begin respiration. As long as all the critical
components for the reaction are available, this cycle provides a constant source of energy for the cell.

Respiration harvests biological energy from fuel molecules, such as carbohydrates, and stores it as ATP. Together with oxygen, the cell converts carbohydrates to carbon dioxide, water and most importantly energy. Respiration is a controlled, multistep process which slowly releases the energy
stored in glucose and converts it to ATP. If all of this energy from glucose
were released at once, most would be lost as heat and light.

Carbohydrates contain high energy bonds that, when broken, release electrons. The first stage, glycolysis, breaks carbohydrates (glucose) into pyruvate molecules. Though the bonds holding pyruvate together contain a
great deal of potential energy, this step yields little energy.

Yeast has been used to make

leavened bread for centuries.
When
yeast
undergoes
fermentation, CO2 is trapped
between gluten and causes
the bread to rise. Ethanol,
another byproduct of yeast
fermentation, generates the
alcohol content in beer, and
the CO2 provides effervescence.
What
ingredients
must be present in order for
this process to occur?

Glycolysis occurs with or without oxygen and takes place in the cytoplasm outside the mitochondria. Interestingly, it is a pathway found in all living things.
C6H12O6 + 6O2 6CO2 + 6H2O + energy
(glucose + oxygen carbon dioxide + water + energy)

Figure 1: Anaerobic vs. aerobic respiration charts.

Aerobic respiration takes place in the mitochondria (a specialized organelle) of

the cell and uses oxygen as the final electron acceptor in the electron transport
pathway. Pyruvate is oxidized to generate energy. Special molecules shuttle
electrons to the ATP production site. Since oxygen has a very high affinity for
electrons, aerobic respiration is the most efficient means of producing ATP (36
per reaction).

Anaerobic respiration takes place in the cytoplasm of the cell and uses other,
less efficient, molecules to transport electrons. If the final transfer molecule is
organic (contains a carbon), the process is called fermentation. Fermentation
is an anaerobic process that reduce regenerate NAD+ so that glycolysis can
continue. Because it cannot fully break down the glucose molecule, fermentation
is far less efficient than aerobic respiration, generating only two ATP molecules.

During physical activity,

cells require more energy.
As long as enough oxygen
can be delivered to cells,
aerobic respiration dominates.
When energy consumption
exceeds the oxygen supply, anaerobic respiration
starts. Lactic acid is a byproduct, and is what causes muscle soreness after a
hard workout!

Yeast cells produce ethanol and CO2 during fermentation. We will measure the production of CO2 to determine the rate of anaerobic respiration in the presence of different carbohydrates.

5 Respirometers (see Figure 2)

Ruler

1% Glucose Solution

Measuring spoon

1% Sucrose Solution

*Warm water

Equal, Splenda, and sugar packets

*Permanent marker

1 Yeast packet

*Stopwatch

(4) 250 mL Beakers

(1) 100 mL Graduated cylinder

*You must provide

Pipettes

Note: Sucrose (a disaccharide) is made up of glucose and fructose. Glucose is a monosaccharide.

Figure 2: Respirometers are created using two test tubes, one small
plastic test tube and one large glass test tube.

1. Completely fill the smallest tube with water and invert the larger tube over it. Push the small tube up (into
the larger tube) until the top connects with the bottom of the inverted tube. Invert the respirometer so that
the larger tube is upright (there should be a small bubble at the top of the internal tube). Repeat this several times as practice strive for the smallest bubble possible. When you feel comfortable with this technique, empty the test tube and continue with this experiment.
2. Mix 1/4 tsp. of yeast into 175 mL of warm (40 - 43C) water in a 250 mL beaker. Stir until dissolved.
Note: Make sure the yeast solution is stirred before each test tube is filled.
3. Label both the big and small test tubes 1-5.
4. In a 250 mL beaker, mix the 1 gram packet of Equal with 100 mL of water. In another 250 mL, mix the
1 gram packet of Splenda with 100 mL of water. In another 250 mL beaker, mix HALF of the 4 gram
packet of sugar with 200 mL of water.
5. Fill the smaller test tubes with 15 mL solution as follows:

Tube 1: 1% glucose solution

Tube 2: 1% sucrose solution

Tube 3: 1% Equal solution

Tube 4: 1% Splenda solution

Tube 5: 1% sugar solution

Note: A good practice in the lab is to rinse the graduated cylinder between each use.
6. Then, fill each tube to the top with the yeast solution.
7. Slide the corresponding larger tube over the small tube and invert it as practiced. This will mix the yeast
and sugar solutions.
8. Place respirometers in the test tube rack, and measure the initial air space in the rounded bottom of the
internal tube. Record these values in the Table 1.
9. Allow the test tubes to sit in a warm place (approximately 37 C) for one hour. A few suggestions are: a
sunny windowsill, atop (not in!) a warm oven heated to 200 F, under a very bright (warm) light, etc.
10. At the end of the respiration period, measure the air space in the internal tubes, and record in Table 1.

Table 1: Gas Production

Tube

Initial Gas Height (mm)

Final Gas Height (mm)

Net Change

1
2
3
4
5

1. Hypothesize why some substances were not metabolized, while others were. Research the chemical formula of Equal and Splenda and explain how it would affect respiration.

2. If you have evidence of respiration, identify the gas that was produced. Suggest two methods for positively identifying this gas.

3. How do the results of this experiment relate to the role yeast plays in baking?

4. What would you expect to see if the yeasts metabolism was slowed down? How could this be done?

5. Indicate sources of error and suggest improvement.

6. Optional: Using the leftover yeast, test some other carbohydrates (e.g. brown sugar, molasses) from your
cupboard to see if the rates of respiration differ. Report your experimental procedure and results below.

We will evaluate respiration in beans by comparing carbon dioxide production between germinated and nongerminated beans. As shown in the balanced equation for cellular respiration, one of the byproducts is CO 2
(carbon dioxide):
C6H12O6 + 6H2O + 6O2 energy + 6CO2 +12H2O
We will use a carbon dioxide indicator (bromothymol blue) to show oxygen is being consumed and carbon
dioxide is being released by the beans. Bromothymol blue is an indicator that turns yellow in acidic conditions,
green when it is neutral and blue when it is in basic conditions. When carbon dioxide dissolves in water,
carbonic acid is formed by the reaction:
H2O + CO2 H2CO3
resulting in the formation of this weak acid. If an indicator such as bromothymol blue is present, what do you
think would happen? (Hint - what color would the indicator change to?)

100 Pinto beans

Parafilm

100 Kidney beans

6 Rubber bands

(6) 250 mL beakers

*Water

6 Measuring cups (small white paper cups)

*Paper towels

Pipette
24 mL Bromothymol blue solution

*You must provide

Figure 3: Beaker set-up

1. Fill two beakers with 200 mL water.

2. Soak 50 pinto beans in Beaker 1P and 50 kidney beans in Beaker 1K for
24 hours.
3. Empty the water from beakers 1P and 1K.
4. Pour the soaked beans onto paper towels, keeping them separated.
5. Label the remaining beakers: Beakers 2P, 3P, Beaker 2K, and 3K.
6. Place several layers of moist paper towels at the bottom of the 250 mL beakers.
7. Place 50 pre-soaked pinto beans into Beaker 1P, 50 control pinto beans in Beaker 2P, and zero beans in
Beaker 3P.
8. Place 50 pre-soaked kidney beans into Beaker 1K, 50 control kidney beans in Beaker 2K, and zero beans
in Beaker 3K.
9. Dispense 4 mL of bromothymol blue solution into each of the three measuring cups, and place the measuring cup inside each beaker (Figure 3).
10. Stretch Parafilm across the top of each beaker. Secure with a rubber band to create an air-tight seal.
Note: If your Parafilm breaks, plastic wrap can also be used.
11. Place the beakers on a shelf or table, and let sit undisturbed at room temperature.
12. Observe the jars at 30 minute intervals for three hours, and record any color change of the bromothymol
blue in Tables 2 and 3.
13. Let the beans and the jar sit overnight. Record your observation in Tables 2 and 3.

Table 2: Bromothymol Blue Color Change Over Time for Pinto Beans
Time

Beaker with Pre-Soaked

Beans

Beaker with Un-Soaked

Beans

Beaker with No Beans

0 min
30 min
60 min
90 min
120 min
150 min
180 min
24 hrs

Table 2: Bromothymol Blue Color Change Over Time for Kidney Beans
Time
0 min
30 min
60 min
90 min
120 min
150 min
180 min
24 hrs

Beaker with Pre-Soaked

Beans

Beaker with Un-Soaked

Beans

Beaker with No Beans

1. What evidence do you have to prove cellular respiration occurred in beans? Explain.

2. Were there differences in the rates of respiration in pinto beans vs. kidney beans? If so, why?

3. If this experiment were conducted at 0C, what difference would you see in the rate of respiration? Why?

4. What is the mechanism driving the bromothymol blue solution color change?

5. What are the controls in this experiment, and what variables do they eliminate? Why is it important to
have a control for this experiment?

6. Would you expect to find CO2 in your breath? Why?

7. What effect would large changes of temperature (e.g., 37 C vs. 45C) have on respiration in beans? Design an experiment to test your hypothesis, complete with controls?

Lab 3

Culturing and Aseptic Technique

Nutrient Broth

Inoculating Microorganisms

Nutrient Agar

Isolation: Streak and Spread Plate Methods

Aseptic Transfer

Microbiologists grow and keep microorganism cultures on media. Media can be in either liquid (broth) or solid
(agar) form. Agar is a complex carbohydrate isolated from seaweed. It has many industrial, food, and scientific uses. It is also a convenient solidifying agent for microbiology media as very few microorganisms can metabolize it. Agar liquefies at 90 - 100 Celsius and solidifies at approximately 42 Celsius.
Regardless of the type, all media must be prepared carefully to include the proper nutrients needed for microbial growth, and, to prevent unwanted growth before the addition of the microbe sample.
Most media is prepared in a sterile procedure, and sterilized
after production. Sterilization of media is commonly performed
using an autoclave that uses pressure and steam to kill any
unwanted organisms, or membrane filtration with positive pressure to filter out any particles above a specific size. Since you
will not have access to these sterilization techniques, the solid
media you use for the experiments in this lab manual will be
prepared sterilely for you. However, you will be required to
melt the media and pour it into Petri dishes in preparation for
growing bacteria. This process must be performed in a manner
that reduces the possibility of introducing unwanted organism Figure 1: The use of agar has greatly aided
many advances in microbiology. It is also used
into the media before being used in the experiment. Additional- in ice cream production.
ly, you will be required to inoculate (introduce a microorganism
to) prepared media plates with samples containing bacteria that you have collected from your environment (or
may be provided to you) and transfer bacteria from one media plate to another, or to other containers. These
procedures must be accomplished without introducing contamination to the original plate, the new plate, the
environment, or yourself. This method is called aseptic technique and is used for almost all procedures involving microbes.

There are numerous types of growth media. Early microbiologists prepared their own media using available materials at the
time, such as extracts of beef and vegetable slices. Robert
Koch (1843-1910) was a German physician/scientist who pioneered many microbiology techniques that are still used today.
Koch was one of the first microbiologists to use a solid form of
media to cultivate microbes. He prepared slices of boiled potatoes and smeared them with samples from diseased animals.
The potato served as a form of growth media for the bacteria
Figure 2: Koch was the first scientist to iso- and using this technique, Koch was able to isolate pure bactelate the causative agent of anthrax, Bacillus
rial cultures that were derived from a single species.
anthracis, using his potato slice method.

Today, media are typically available in powdered form and only need to be rehydrated. All complete growth media need to provide the nutrient requirements of the diverse
bacteria grown on them. These nutrients must include a carbon source (which can also supply energy) and a
nitrogen source. Both of these nutrients can be supplied by plant or animal sources. Additional nutrients include phosphorus, sulfur, minerals, and water. Media made from these sources are called undefined or complex media since the exact makeup and amount of carbon and nitrogen are not known. Defined media is that
in which each chemical component and its amount is precisely known and controlled to facilitate the growth
of specific microbes. Various media are also used to enrich or select for particular types of bacterial growth;
these are called selective media, which can be complex or defined.
Aseptic technique is used when adding bacterial samples to media
plates and also when moving bacterial colonies from one plate to another. As mentioned above, aseptic techniques minimize the likelihood of contaminating the pure bacterial samples with unwanted microbes from the surrounding environment, or those that normally inhabit our bodies. Aseptic techniques also are important to reduce the
spread of cultured bacteria (which may be potentially hazardous) to
our surroundings, thereby limiting or preventing exposure to pathogenic agents. In a microbiology lab, scientists can sterilize instruments with heat (Bunsen burner), with an autoclave (high pressure
and temperature), or by gas sterilization. Throughout the experiments
in this lab kit, you will use sterile, disposable instruments or a candle
(for sterilization) because you do not have access to lab-grade sterilization equipment. You will also need to use proper aseptic technique
to ensure the purity of your cultures. The following list provides some
common aseptic techniques used in the microbiology lab:

Figure 3: Different microorganisms colonize in different shapes and colors.

The streaking (also referred to as plating) method may also influence a colonys visual appearance.

Reduce the potential to contaminate your surroundings by working in a clean, uncluttered space.

Be organized and read through each experiment thoroughly before starting. Gather and prepare
all necessary reagents and instruments before beginning the experiments.

Immediately cover plates after pouring media into them. When opening a plate to either inoculate it or to transfer bacteria from it, use the lid as a shield to limit contamination from airborne
sources.

When using tubes containing media or other liquids, hold the tube at an angle and immediately
recap to limit contamination from airborne sources.

Always use a new, clean, sterile, disposable loop or transfer pipette when transferring multiple
samples or colonies of bacteria, unless otherwise directed in the experiment instructions.

Store prepared, solidified agar plates (not yet inoculated) upside-down so that any condensation
that forms on them will not drop onto the media surface.

Figure 4: Chlorine bleach is an accepted

liquid for sterilization.

Figure 5: Agar color can vary based on the ingredients. It is often clear-yellow or red.

Disinfect all cultures at the end of every experiment (unless otherwise instructed) by adding a
10% bleach solution to the culture plates and letting them sit at room temperature for 20 minutes.
Household bleach contains 5.25% sodium hypochlorite, and is corrosive so surfaces should be
wiped with water after using bleach. A 10% solution of household bleach will kill many organisms
immediately, but may take up to 20 minutes for full sterilization.

After flooding the plates, pour the bleach down a drain, seal the plates with Parafilm, and dispose of them in the trash.

Be sure to clean and disinfect your work area with disinfecting wipes or 70% ethanol after each
experiment.

Obtaining pure bacterial cultures is important for the microbiologist as this greatly facilitates identifying what
types of bacteria occur in a mixed population. Frequently, a dilution series of the sample is prepared such
that the initial number of bacteria is reduced so that when the sample is plated onto growth media, individual
bacteria are separated from one another. These individually isolated bacteria cells then produce a colony of
cells, all derived from the parent bacteria. Colonies arising from different types of bacteria often exhibit
unique morphologies that can aid in identifying the original bacteria. In the experiments outlined in this manual, be sure to always note the appearance (morphology, color, edges, texture, pattern of growth) of the bacterial colonies that grow on your plates.

Growth media is used to grow and maintain microorganisms. In this experiment, you will aseptically prepare
nutrient agar plates and inoculate microorganisms with bacterial samples obtained from you and your environment.

4 Sterile cotton swabs

*Microwave or boiling water bath

(5) 5 cm. Petri dishes

*10% Bleach solution

Nutrient agar

*Permanent marker

Parafilm
1 Pair of gloves
Hot pad

*You must provide

1. Loosen or remove the cap on the Nutrient agar bottle. Place the bottle in the microwave (if you do not
have a microwave, place the bottle in a heat-safe bowl and pour boiling water around the bottle) and heat
until the entire agar bottle is liquefied. You will need to remove the bottle and swirl every 10 seconds to
distribute the heat.
Note: The bottle may explode, causing agar to project into your microwave if the bottle is not removed
from heat every 10 seconds. It is very important to loosen or remove the cap to avoid creating a high
pressure environment. If you notice the agar boiling over, STOP the microwave and let the bottle cool
down before handling. Hot agar can violently explode out of the bottle if heated too quickly and/or
shaken. After boiling has stopped, use a hot pad protecting your hands and remove the bottle from the
microwave. Use caution when removing the bottle from the microwave as it will be HOT!
2. Gently swirl the bottle to mix the solution a final time.
3. Slowly pour the liquefied agar solution into the bottom half of five petri dishes so that it covers the entire
bottom of the dish. It is important that the entire bottom is coated and that the agar is given time to spread
out over the dish.
4. Place the lids onto the petri dishes and let them sit at room temperature until the plates are solidified and
cooled. The plates can be used immediately or stored in the refrigerator for later use.
5. If you have stored the plates in the refrigerator, remove them and allow them to sit at room temperature
for at least one hour before use.

6. Look around for three surfaces to swab microorganisms from. Things such as shoes, a table, teeth, mouth
or throat, bathroom doors, and shopping carts can be great sources. Try to avoid surfaces that are frequently cleaned with antibacterial detergents and soaps.
7. Use a permanent marker to label the bottom of one of your petri dishes with the title of the surface you
select (e.g., if you choose a table, label the dish as Table).
8. Put on a pair of gloves, and begin collecting your samples by rubbing a cotton swab on the location you
select. Be sure to get good coverage on the cotton swab; and, use a new cotton swab for every sample.
Note: It helps if the cotton swab has been lightly moistened with sterile water prior to collecting your
samples. The extra water helps promote visible, well-developed bacterial growth.
9. Remove the lid from the agar plate but hold it closely over the top of the plate to use as a shield to prevent
airborne contamination of the plate.
10. Carefully streak the cotton swab onto the gelled medium (agar). To do this, start at the top and work down

in a zigzag motion. Ensure that you do not press too hard when streaking the swab on the agar to prevent cutting into the agar surface.
11. Place the lid onto the agar plate and seal it with a strip of Parafilm (hold one end of the Parafilm
firmly against the side of the petri dish and stretch the other side to cover the entire perimeter).
12. Place the plate upside down in a warm area to incubate.
13. Repeat steps 6 - 11 for two additional surface areas and agar plates.
14. Use a permanent marker to label the bottom of the fourth petri dish Control. Take a sterile cotton
swab and rub it onto the control agar plate. Do not rub your sterile cotton swab on any surface. This will
test for your accuracy in keeping the plates and swabs sterile while performing the experiment.
15. Repeat steps 10 - 11 on the Control petri dish.
16. Use a permanent marker to label the fifth petri dish Airborne Contamination. Remove the lid from the
plate and expose the agar to the air for 1 hour. It may be helpful to place the dish close to an airregister or fan to increase the air circulation surrounding the dish.
17. Repeat Step 11 for the Airborne Contamination petri dish.
18. Let all five of the plates incubate in a warm area for 3 days and then observe the growth. Record their
growth in Table 1.
**Answer questions 1 - 3 in the Post-Lab after you have recorded your growth observations.**
19. Set the plate with the most bacterial growth aside for the next experiment.
20. Pour approximately 5 mL of the 10% bleach solution onto the surface of the remaining agar plates, allowing it to cover the entire surface. Incubate them for 20 minutes, and then pour the bleach down the
sink with running water.
21. Seal the petri dishes with Parafilm and dispose of them in the trash.

Table 1: Experiment 1 Colony Growth

Growth (color, amount,
shape, etc.)

Plate #

Condition

Surface 1

Surface 2 door handel

Surface 3

Control

Airborne Contamination

teeth

stove top

very little growth, a tiny bit of white

bacteria
very little growth, a tiny bit of white
bacteria
showed lots of bacteria, it spread
over the entire center of dish
showed no bacteria
had a small amount of bacteria
around the edge of dish

1. Draw the colonies that grew on your plates, indicating the number of different colonies and identifying
their morphology, color, edges, texture, and pattern of growth.
2. Which plate grew the most bacterial species (based on number of different colonies)? Was this a surprise? Why or why not?

The stove top plate grew the most bacteria, considering that the stove top is very dirty it was not surprising.

3. Was the control plate (plate #4) free of bacterial colonies? If not, what could be the source of the contamination? It was free of bacteria
4. Did the airborne contamination plate (plate #5) contain any growth? Were any of the colonies similar in
appearance to those found on the other plates? How does this plate demonstrate the need for following
aseptic technique when culturing bacteria? The airborne did have bacteria, mainly little blotches around the rim of the plate, they were
similar to the other plates.

Transferring an isolated bacterial colony from one plate to another or to a different type of growth surface is
an important technique in microbiology. Frequently, a microbiologist is required to initially grow bacteria on
one type of media then transfer it to a selective and/or differential plate to further characterize the bacteria.
Stab tubes can be used for testing the motility of a microbe or for longer term storage at refrigerator temperature (4 Celsius). Again, this transfer must be performed using aseptic technique to ensure the purity of the
originally isolated microbe.

4 Sterile disposable snap-cap tubes

Hot pad

(4) 5 cm. Petri dishes

100 mL Beaker

Nutrient agar

*Microwave or boiling water bath

Parafilm

*10% Bleach solution

1 Pair of gloves

*Permanent marker

6 Inoculating loops
1 Petri dish with bacteria growth from Experiment 1 *You must provide

1. Prepare four Nutrient agar plates by following Steps 1 - 5 of Experiment 1.

2. Slowly pour agar into the snap-cap tubes until each snap-cap tube contains approximately 2 mL of agar.
The snap-cap has a 5 mL volume capacity; therefore, your snap-cap should be approximately 40% full.
Note: The snap-caps tubes are referred to as stab tubes after the agar has been poured and
gelled.
3. Snap the lid on the caps and store them upright at room temperature until the agar has solidified and
cooled. The tubes can be used immediately or stored at 4 C. If stored, let warm to room temperature for
approximately 1 hour before use.
Note: Stab tubes should be vertically positioned in a 100 mL beaker to allow the agar to solidify
properly.
3. Put on a pair of gloves, and find the petri plate that you reserved from Experiment 1. Locate three separate bacterial colonies. If possible, these colonies should have different morphologies from each other.
Record observations of their physical characteristics in Table 2.
4. Remove about half of 1 of the selected colonies on the plate by scraping it up with an inoculating loop.

5. Remove the lid from one of the agar plates prepared in Step 1. Remember to hold the lid closely over the
top of the plate to prevent airborne contamination of the plate.
6. Gently streak an inoculating loop over the surface of the agar in a zig-zag pattern, taking care to not dig
into the surface of the agar with the loop. Dispose of the loop.
7. Place the lid on the plate and seal it with Parafilm.
8. Turn the plate upside-down and label it as Plate #1 with your permanent marker.
9. Use a new inoculating loop to remove the other half of the previously selected colony.
10. Stab/push the loop approximately half way into one of the stab tubes.
11. Pull the inoculating loop straight up and out through the same path that it went down into the media. Dispose of the loop.
12. Snap the lid on the tube, and label the tube as Tube #1 with your permanent marker. Be sure to press
the lid all the way down to create an air-tight seal.
13. Repeat Steps 6 - 9 and 10 - 13 with two other bacterial colonies from your reserved plate. Label the
plates and tubes as Plate #2 Tube #2, Plate #3, and Tube #3, respectively.
14. Label the fourth tube and plate as Control. Do not streak or stab anything onto/into them. Leave these
containers covered.
15. Incubate the plates and tubes at room temperature or warmer (not to exceed 37.7 C or 100 F) for 3
days and observe for growth. Record your results in the Table 3.
16. Pour approximately 5 mL of the 10% bleach solution onto the surface of the remaining agar plates, allowing it to cover the entire surface. Incubate them for 20 minutes, and then pour the bleach down the
sink with running water.
17. Seal the petri dishes with Parafilm and dispose of them in the trash.
18. Fill the stab tubes with the 10% bleach solution and incubate them for 20 minutes at room temperature.
19. Pour the bleach down a drain with running water, snap the caps onto the tubes, and dispose of them in
the trash.

Table 2: Initial Reserved Plate Colony Growth Observations

Plate Sample
1
2
3

Appearance (morphology, etc.)

Table 3: Final Plate and Stab Tube Growth Observations

Sample

Form

Growth? (Yes or No)

Same Appearance
as Initial Plate?
(Yes or No)

Effective Transfer?
(Yes or No)

Plate
1
Stab Tube
Plate
2
Stab Tube
Plate
3
Stab Tube
Plate
Control
Stab Tube

1. Did the colonies that grew on the transfer plates retain the same appearance from the initial plate? Why
or why not?
2. Were the colonies uniform in appearance on the transfer? What could account for any differences seen?
3. Were you able to detect growth in the stab tubes?
4. Did the growth occur throughout the depth of the stab? If not, what could account for lack of bacterial
growth?
5. What was the purpose of the controls? What type of control is this: positive or negative?

Good Laboratory Techniques

Science labs, whether at universities or in your home, are places of adventure and discovery. One of the
first things scientists learn is how exciting experiments can be. However, they must also realize science
can be dangerous without some instruction on good laboratory practices.

Read the protocol thoroughly before starting any new experiment.

You should be familiar with the action required every step of the
way.

Keep all work spaces free from clutter and dirty dishes.
Read the labels on all chemicals, and note the chemical safety rating on each container. Read all Material Safety Data Sheets
(provided on www.eScienceLabs.com).

Thoroughly rinse labware (test tubes, beakers, etc.) between experiments. To do so, wash with a soap and hot water solution using a bottle brush to scrub. Rinse completely at least four times.
Let air dry

Use a new pipet for each chemical dispensed.

Figure 1: A benchcoat will

prevent any spilled liquids
Wipe up any chemical spills immediately. Check MSDSs for special
from contaminating the surhandling instructions (provided on www.eScienceLabs.com).
face you work on.

Use test tube caps or stoppers to cover test tubes when shaking or
mixing not your finger!

Figure 2: Special measuring tools in make experimentation easier and more accurate in the lab. A shows a beaker, B graduated cylinders, and C test tubes in a test
tube rack.

When preparing a solution, refer to a protocol for any specific

instructions on preparation. Weigh out the desired amount of
chemicals, and transfer to a beaker or graduated cylinder.
Add LESS than the required amount of water. Swirl or stir to
dissolve the chemical (you can also pour the solution back
and forth between two test tubes), and once dissolved, transfer to a graduated cylinder and add the required amount of
liquid to achieve the final volume.

A molar solution is one in which one liter (1L) of solution contains the number of grams equal to its molecular weight.
For example:
1M = 110 g CaCl x 110 g CaCl/mol CaCl
(The formula weight of CaCl is 110 g/mol)

Figure 3: Disposable pipettes aid in accurate measuring of small volumes of

liquids. It is important to use a new pipette for each chemical to avoid contamination.

A percent solution can be prepared by percentage of weight of chemical to 100ml of solvent (w/v) ,
or volume of chemical in 100ml of solvent (v/v).
For example:
20 g NaCl + 80 mL H2O = 20% w/v NaCl solution

Concentrated solutions, such as 10X, or ten times the normal strength, are diluted such that the final
concentration of the solution is 1X.
For example:
To make a 100 mL solution of 1X TBE from a 10X solution:
10 mL 10X TBE + 90 mL water = 100ml 1X TBE

Always read the MSDS before disposing of a chemical to insure it does not require extra measures.
(provided on www.eScienceLabs.com)

Avoid prolonged exposure of chemicals to direct sunlight and extreme temperatures. Immediately
secure the lid of a chemical after use.

Prepare a dilution using the following equation:

Where c1 is the concentration of the original solution, v1 is the volume of the original solution,
and c2 and v2 are the corresponding concentration and volume of the final solution. Since you
know c1, c2, and v2, you solve for v1 to figure out how much of the original solution is needed to
make a certain volume of a diluted concentration.

If you are ever required to smell a chemical, always waft a gas toward you, as shown in the
figure below.. This means to wave your hand over the chemical towards you. Never directly
smell a chemical. Never smell a gas that is toxic or otherwise dangerous.

Use only the chemicals needed for the activity.

Keep lids closed when a chemical is not being used.

When diluting an acid, always slowly pour the acid into the water. Never pour water into an acid, as this could cause both splashing and/or an explosion.

Never return excess chemical back to the original bottle. This can contaminate the chemical
supply.

Be careful not to interchange lids between different chemical bottles.

When pouring a chemical, always hold the lid of the chemical bottle between your fingers. Never lay the lid down on a surface. This can contaminate the chemical supply.

When using knives or blades, always cut away from yourself.

Wash your hands after each experiment.

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